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J Comp Physiol A (2014) 200:527–536
DOI 10.1007/s00359-014-0892-4
ORIGINAL PAPER
Physiological basis of phototaxis to near‑infrared light
in Nephotettix cincticeps
Motohiro Wakakuwa · Finlay Stewart · Yukiko Matsumoto ·
Shigeru Matsunaga · Kentaro Arikawa
Received: 15 January 2014 / Revised: 13 February 2014 / Accepted: 14 February 2014 / Published online: 11 March 2014
© Springer-Verlag Berlin Heidelberg 2014
Keywords Visual pigment · Photoreceptor · Ommatidia ·
Spectral sensitivity · Phototaxis · Hemipteran insect
Introduction
Ever since Karl von Frisch demonstrated color vision in
honeybees (Frisch 1914), its neuronal basis has been a
major theme in the field of behavioral neuroscience (Gold-
smith 1961; Menzel 1979; Briscoe and Chittka 2001; Kel-
ber 2006). One of the conspicuous features of honeybee
color vision is that it extends into the ultraviolet (UV),
while they cannot discriminate red from gray (Bertholf
1931a, b). This shifted visible spectrum in honeybees was
firmly established by electrophysiological identification
of the UV, blue (B) and green (G) receptors in the com-
pound eye (Autrum and von Zwehl 1964; Autrum 1968).
However, color vision performance in insects appeared
to be variable among species: for example, butterflies—
unlike honeybees—can readily discriminate red from other
colors (Ilse 1928, 1937). Therefore, considerable attention
was given to the question of whether or not insects possess
“red” receptors (Goldsmith 1965).
The existence of red receptors was first suggested in but-
terflies by electroretinogram recordings (Swihart and Gordon
1971; Eguchi et al. 1982). Red-absorbing visual pigments
were also identified in butterflies and moths by microspec-
trophotometry (Bernard 1979; Langer et al. 1979), but the
conclusive demonstration of red receptors was obtained by
intracellular recording of single photoreceptor cells in butter-
flies (Matic 1983; Arikawa et al. 1987a; Shimohigashi and
Tominaga 1991). Red receptors were also subsequently iden-
tified in dragonflies (Yang and Osorio 1991), wasps (Peitsch
et al. 1992) and, recently, in a flower-visiting beetle (Mar-
tinez-Harms et al. 2012) and a damselfly (Henze et al. 2013).
Abstract In a previous study of the phototaxis of green
rice leafhoppers, Nephotettix cincticeps (Hemiptera,
Cicadellidae), we found positive responses to 735 nm light.
Here, we investigated the mechanism underlying this sen-
sitivity to near-infrared light. We first measured the action
spectrum using a Y-maze with monochromatic lights from
480 to 740 nm. We thus found that the action spectrum
peaks at 520 nm in the tested wavelength range, but that
a significant effect is still observed at 740 nm, albeit with
a sensitivity 5 log units lower than the peak. Second, we
measured the spectral sensitivity of the eye, and found that
the sensitivity in the long-wavelength region parallels the
behaviorally determined action spectrum. We further iden-
tified mRNAs encoding opsins of ultraviolet, blue, and
green-absorbing visual pigments, and localized the mRNAs
in the ommatidia by in situ hybridization. The electro-
physiology, molecular biology and the anatomy of the
eye together indicate that the eyes of N. cincticeps do not
contain true “red” receptors, but rather that the behavioral
response to near-infrared light is mediated by the tail sensi-
tivity of the green receptors in the long-wavelength region
of the spectrum.
M. Wakakuwa · F. Stewart · K. Arikawa (*)
Laboratory of Neuroethology, Sokendai-Hayama (The Graduate
University for Advanced Studies), Shonan Village, Hayama,
Kanagawa 240-0193, Japan
e-mail: arikawa@soken.ac.jp
Y. Matsumoto
National Institute of Agrobiological Sciences, Owashi 1-2,
Tsukuba, Ibaraki 305-8634, Japan
S. Matsunaga
Central Research Laboratory, Hamamatsu Photonics K.K., 5000,
Hirakuchi, Hamakita-ku, Hamamatsu, Shizuoka 434-8601, Japan
528 J Comp Physiol A (2014) 200:527–536
1 3
Two mechanisms underlying the spectral sensitivity of
red receptors have been identified. The first mechanism is
straightforward, involving the acquisition of a new visual
pigment opsin through gene duplication events. In Papilio
butterflies, for example, red receptors are found to express
a visual pigment with peak absorption at 575 nm (Kita-
moto et al. 1998; Briscoe 2000). The second mechanism
is widely found in pierid butterflies, in which short wave-
length-absorbing screening pigments shift the effective
absorption spectrum of the visual pigments to considerably
longer wavelengths. For instance, the Eastern clouded yel-
low, Colias erate, has receptors with visual pigment absorb-
ing maximally at 565 nm. Screening pigment strongly
filters the blue-green range of incoming light, shifting the
spectral sensitivity toward red. The resulting peak sensitiv-
ity, 660 nm, is the longest peak wavelength so far identified
among insect photoreceptors (Ogawa et al. 2013).
While the spectral sensitivities of the visual photorecep-
tors of Lepidoptera and Hymenoptera have been extensively
investigated, studies in other insect orders have remained
sparse. How common is red sensitivity among insects? Are
there any other mechanisms for producing red receptors? In
the course of studying the light responses of leafhoppers and
planthoppers (order Hemiptera, suborder Auchenorrhyncha),
we observed positive phototaxis to 735 nm light (Matsumoto
et al. 2014). This near-infrared (IR) light is almost invis-
ible even to humans, and was thought to be totally invisible
to insects. Probably due to this prevailing view, this wave-
length region has never been tested as far as we know. The
possibility that leafhoppers have some specific mechanism
for detecting near IR light based on previously unidentified
molecular and/or optical mechanisms motivated us to initiate
a detailed study of this Hemipteran insect. Among Hemip-
teran species, the visual system of the common backswim-
mer, Notonecta glauca (order Hemiptera, suborder Heterop-
tera), has been extensively studied, but their eyes appear to
be furnished only with UV, B and G receptors, as in hon-
eybees (Bruckmoser 1968; Schwind et al. 1984). To address
the question of whether the leafhoppers have specific pho-
toreceptors that are sensitive to near IR light, we studied the
spectral organization of their eyes by combining electrophys-
iology, molecular biology and anatomical methods. We also
measured the action spectrum of the phototactic response,
and found that the near IR sensitivity must be attributed to
the sensitivity of green receptors in the compound eye.
Materials and methods
Animals
We used male green rice leafhoppers, Nephotettix cincti-
ceps, obtained from a laboratory stock culture maintained
in the National Institute of Agrobiological Sciences,
Tsukuba, Japan. The individuals were reared on rice seed-
lings at 25 °C under a light regime of 16 h light:8 h dark.
Behavioral analysis
Behavioral experiments were carried out in a dark room at
25 °C using a Y-maze apparatus made of transparent acrylic
plates (Fig. 1a, b). The walls of the Y-maze were covered
with black cardboard. The Y-maze was illuminated from
below using a 180 mm × 150 mm LED panel emitting IR
light peaking at 940 nm (TMN-4, Aitec, Yokohama Japan),
which was set on a rotation stage (120-B, Meiritsu, Yoko-
hama, Japan) (Fig. 1b). An IR-sensitive video camera (WAT-
221S, Watec, Yamagata, Japan) equipped with a wide-angle
lens (Zuiko 16 mm, Olympus, Tokyo, Japan) recorded the
movement of insects in the Y-maze from above.
We define four areas in the Y-maze: the starting area, the
selection area, and two arms (Fig. 1a). The border between
the starting and the selection areas could be closed with a
black screen. The end walls of the arms were fitted with
frosted screens. The end of one arm was illuminated with
monochromatic light from outside, while the other was
kept dark (Fig. 1c). Monochromatic stimuli were provided
by a 100 W xenon arc through a series of narrow-band
interference filters, with peak transmittance at 480, 520,
590, 660, 700, 720, and 740 nm (Asahi Spectra, full width
at half maximum (FWHM) 10–14 nm). The intensity was
varied over a range of 7 log units. The photon flux of each
monochromatic light was measured at the inner surface of
the frosted screen using a radiometer (model 470D; Sanso,
Tokyo, Japan). The illuminated arm was switched after
every two tests by rotating the Y-maze with the LED panel.
For each illumination condition with a certain wave-
length and intensity, at least 20 individuals were used.
Each test was carried out between 5 and 11 individuals
in the Y-maze simultaneously. Before commencing a test,
the starting and selection areas were separated by a black
screen. Leafhoppers were released into the starting area
and kept for 5 min in the dark, i.e., without the monochro-
matic stimulus; the 940 nm illumination for the recording
was continuously on. The video camera and stimulus light
were turned on after the initial 5 min, and the screen was
removed. The movements of the leafhoppers were then
recorded for a period of 2.5 min at a sampling frequency of
30 frames/s, which enabled unambiguous identification of
each individual in the image data.
Figure 1c shows sample trajectories of ten leafhoppers
in the Y-maze. After tracing the leafhoppers’ trajectories,
their phototactic behavior was quantified as follows. For
each frame, each individual was given a score, S, accord-
ing to its location in the Y-maze (Fig. 1c): +1 if inside the
illuminated arm, −1 for the un-illuminated arm, and 0 for
529J Comp Physiol A (2014) 200:527–536
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the starting and the selection areas. The attraction index of
individual i, Ai, was calculated by:
Ai=
n
j=1Si,j
n,
where Si,j is the score of individual i in frame number j, and
n is the total number of frames. If an insect quickly moves
into the illuminated arm and stays there until the test ends,
the A value for that individual would be very close to +1.
If an insect does not move from the starting area or moves
randomly in the Y-maze, the A value would be zero. The
statistical significance of the attraction index values was
tested using the Mann–Whitney U test versus the values
measured in the dark as control.
Electroretinography
The spectral sensitivity of the leafhopper’s compound eye
was determined by recording electroretinograms (ERG)
induced by monochromatic stimuli provided by a 500 W
xenon arc lamp through one of a series of narrow-band
interference filters ranging from 300 to 740 nm (Asahi
Spectra, FWHM 10–14 nm). The light beam was focused
on the tip of an optical fiber whose other end was placed
close to the compound eye. The quantum flux of each
monochromatic stimulus was measured using a radiom-
eter (model-470D, Sanso, Tokyo, Japan) and adjusted to a
standard number of photons using an optical wedge.
A leafhopper was fixed with beeswax onto a plastic
stage and mounted in the recording chamber. A chloridized
silver wire inserted into the abdomen served as the refer-
ence electrode. The tip of a glass micropipette filled with
tap water touched a small amount of conductive paste at the
eye surface. The ERG was recorded through a MEZ-7200
preamplifier (Nihon Kohden, Tokyo, Japan) connected to
a computer via a MP-150 AD converter (BIOPAC, USA).
After leaving the sample for 10 min in the dark, the eye
was stimulated with a series of monochromatic flashes
of 100 ms duration, spaced 5 s apart, to record spectral
responses. The wavelength was first swept from short to
long wavelengths, and then the procedure was repeated in
the reverse direction. Such pairs of bidirectional record-
ings were typically repeated five times yielding ten spectral
scans. The response–stimulus intensity (V − log I) func-
tion was also recorded over a 4-log unit intensity range at
several wavelengths. The V − log I data were fitted to the
Naka–Rushton function, V/Vmax = In/(In + Kn), where I is
the stimulus intensity, V is the response amplitude, Vmax is
the maximum response amplitude, K is the stimulus inten-
sity eliciting 50 % of Vmax, and n is the exponential slope.
We then converted the spectral response into spectral sen-
sitivity, which is the reciprocal of the stimulus intensity
required for a criterion response.
Histology
The cellular organization of the ommatidia was stud-
ied using light and electron microscopy during the light
Fig. 1 Y-maze apparatus. a Top view. The apparatus is made of
acrylic plate, and the sidewalls are covered with black cardboard both
inside and outside. The length, width and the depth of the arms and
the starting area are 50, 45 and 25 mm, respectively. b Side view. The
Y-maze is illuminated from below with a 940 nm-emitting LED panel
set on a rotation stage. c Trajectories of ten individuals. Colors of tra-
jectories correspond to different individuals. Stimulus light is deliv-
ered from the left arm. Numbers (+1, −1, 0) are the scores given to
individuals that were in the respective areas to calculate A values
530 J Comp Physiol A (2014) 200:527–536
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period, i.e., the insects were moderately light adapted.
Bisected head tissues were prefixed in 2.5 % glutaralde-
hyde and 2 % paraformaldehyde in 0.1 M sodium caco-
dylate buffer (CB, pH 7.4) for 2 h at room temperature,
and then postfixed in 2 % OsO4 in 0.1 M CB. After being
dehydrated in an acetone series, the tissues were embed-
ded in Quetol 812 (Nisshin EM, Tokyo, Japan). Semi-thin
sections were stained with Azur II for light microscopy,
while ultrathin sections for electron microscopy were
double stained with uranyl acetate and lead citrate, and
observed in a H-7650 transmission electron microscope
(Hitachi, Tokyo, Japan).
Molecular cloning
Total RNA was extracted from compound eyes using RNe-
asy (Qiagen KK, Tokyo, Japan). To amplify fragments
of cDNA potentially encoding opsins of the UV, B and L
classes, we carried out RT-PCR with degenerate prim-
ers designed based on consensus sequences of arthropod
opsins so far identified. Full-length cDNAs were obtained
by 5′- and 3′-RACE method. Amplified fragments were
purified, cloned using pGEM-T vector (Promega, Madison,
USA) and sequenced using Big Dye Terminator v3.1 with
a DNA analyzer (model 3700, Applied Biosystems, War-
rington, UK). The obtained sequences were aligned with
other opsins and processed for phylogenetic analysis by
the neighbor-joining (NJ) method using the MEGA 5.2.2
software (Tamura et al. 2007). The reliability was based on
1,000 bootstrap replicates.
Double-targeted in situ hybridization
To localize opsin mRNAs in the retina, we carried out dou-
ble-targeted in situ hybridization. Two opsin mRNAs were
simultaneously detected using a mixture of a digoxigenin-
labeled and a biotin-labeled cRNA probe, each specific to
different opsin mRNAs. The probes were synthesized from
linearized plasmid carrying partial sequences of identified
opsin mRNA by in vitro transcription.
Bisected heads of N. cincticeps were fixed in 4 % para-
formaldehyde in 0.1 M sodium phosphate buffer (pH 7.2)
and then embedded in paraffin. The paraffin-embedded
tissues were sectioned at about 6 μm thickness with a
rotary microtome. The sections were deparaffinized and
treated at 45 °C with hybridization solution (300 mM
NaCl, 2.5 mM EDTA, 200 mM Tris–HCl, pH 8.0, 50 %
formamide, 10 % dextran sulfate, 1 mg/ml yeast tRNA,
and 1× Denhardt’s solution) containing 0.5 μg/ml of
cRNA probes. After being washed with 50 % formamide-
2X SSC at 55 °C, the hybridized digoxigenin-labeled
probe was detected using anti-digoxigenin antibody and
then visualized using 4-nitroblue-tetrazolium chloride and
5-bromo-4-chloro-3-indolyl phosphate. The sections were
briefly washed and treated with 100 mM glycine solution
(pH2.2) to remove anti-digoxigenin antibody. Hybridized
biotin-labeled probe was detected using streptavidin-conju-
gated alkaline phosphatase and visualized using Fast Red.
Results
Behavioral experiments
We investigated the phototactic sensitivity of leafhop-
pers to monochromatic light using a Y-maze apparatus.
By way of example, Fig. 1c shows the trajectories of ten
individuals responding to 480 nm light at an intensity of
3.79 × 1013 photons m−2 s−1. The mean attraction index
(A) of these ten individuals was 0.874.
Fig. 2 Phototactic behavior of N. cincticeps under seven monochro-
matic lights. a Relation between the attraction index (A) and the
intensity of the stimulus light. Mean ± standard error. Solid curves
are visually fitted sigmoidal curves with the maximal value at A = 1.
The black dotted line is the control A value without stimulus light.
Asterisks indicate A values differing significantly from the dark con-
trol (P < 0.05, Mann–Whitney U test). b Action spectrum
531J Comp Physiol A (2014) 200:527–536
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We measured A values for seven wavelengths at vari-
ous intensities (Fig. 2a). As a control, we recorded the
movement of leafhoppers in the Y-maze in the “dark”, i.e.,
without any monochromatic stimuli in the arms and illumi-
nated only with 940 nm light from below. The leafhoppers
became rather inactive and moved randomly, so that the
resulting average A index in the dark was practically zero
(−0.0082 ± 0.120894, dashed line in Fig. 2a).
At all wavelengths tested, leafhoppers were positively
phototactic, i.e., the attractiveness of the stimuli increased
with the light intensity. For instance at 480 nm, the mini-
mum light intensity required to elicit positive phototaxis
was about 1.2 × 1012 photons m−2 s−1. Saturation occurred
at ~1.2 × 1014 photons m−2 s−1. The animals’ phototactic
sensitivity decreased as the wavelength became longer.
Surprisingly, the leafhoppers were attracted even to 740 nm
light; the A index at 740 nm was significantly higher than
that of the dark control, provided the light intensity was
sufficiently high. Assuming that at all wavelengths all indi-
viduals are attracted to the illuminated arm under sufficient
intensity of stimulus, we visually fitted a sigmoidal curve
with the maximum at A = 1 to each set of data. To obtain
the action spectrum of phototactic behavior, we plotted the
photon flux required for eliciting A = 0.5 versus wave-
length using the sigmoidal curves (Fig. 2b).
To investigate whether the phototaxis is mediated by the
compound eyes, we occluded the compound eyes of five
individuals with black paint. Even when the animals were
released in the starting area with 520 nm monochromatic
light at the highest intensity, none moved out from the start-
ing or selection area within the 2.5 min recording period,
meaning that for this control experiment A = 0.0. In other
words, the compound eyes are essential for the leafhopper’s
phototaxis behavior.
Spectral sensitivity of the compound eye of N. cincticeps
We measured the spectral sensitivities of the compound
eye in six adult leafhoppers, three males and three females
(Fig. 3a). The sensitivity curves, with a primary peak at
520 nm and a secondary peak at 360 nm, could be well
fitted by a sum of the absorption spectra of three putative
visual pigments (Govardovskii et al. 2000) with absorp-
tion maxima at 354, 449 and 527 nm and with ratio of the
amplitudes 1:0.55:6.52, respectively. This suggests that the
eyes are furnished with UV, B and G receptors. No signifi-
cant sexual difference was detected in the spectral sensitiv-
ity (Fig. 3a).
Figure 3b shows the averaged spectral sensitivities of
both sexes on a logarithmic scale, superimposed with the
action spectrum shown in Fig. 2b. Neither the spectral sen-
sitivity nor the action spectrum shows conspicuous peaks
and/or shoulders in the long-wavelength region.
Rhabdom organization and expression pattern of three
opsin mRNAs
We identified and cloned three cDNAs encoding visual
pigment opsins from RNA extracted from the compound
eye. Phylogenetic analysis using the neighbor-joining
method revealed that one of these cDNA sequences
clustered in each of the UV, blue, and long-wavelength
absorbing opsin clades of insects (Fig. 4). We thus
termed the opsins N. cincticeps UV (NcUV), NcB and
NcL, respectively.
We localized the mRNAs encoding these opsins in the
retina by in situ hybridization. Prior to the in situ hybridi-
zation analysis, we investigated the cellular organization
of the ommatidia of N. cincticeps. Figure 5a is a light
Fig. 3 Spectral sensitivity of the compound eye of N. cincti-
ceps determined by ERG. a Spectral sensitivities of males (open
circles) and females (filled circles). Dotted color lines show the
absorption spectra of three visual pigments peaking at 354, 449
and 527 nm based on a template (Govardovskii et al. 2000). The
solid red line is the weighted summation of three spectra with ratio
UV:B:G = 1:0.55:6.52, normalized at 527 nm. b Averaged spectral
sensitivity (open circles) and the action spectrum (dashed line) super-
imposed, both normalized at 520 nm. Red line indicates the summa-
tion of three visual pigment spectra normalized at 527 nm
532 J Comp Physiol A (2014) 200:527–536
1 3
micrograph of a semi-thin transverse section through the
retina of a light adapted insect, stained with Azur II. Each
ommatidium contains many pigment granules surround-
ing the rhabdom. The pigment granules are located about
1 μm outside the edge of the rhabdom, whose transverse
section is circular and about 2 μm in diameter (Fig. 5b).
Because of this distance, the pigment granules should have
little effect in absorbing the boundary wave, i.e., the light
propagating outside the rhabdom. In addition, the pig-
ment is black, as it is in the secondary pigment cells of the
eye of Colias erate (Arikawa et al. 2009). The rhabdom
is composed of the rhabdomeres of eight photoreceptor
cells, which all contribute straight and parallel microvilli
throughout the entire length of the ommatidia, i.e., there are
no clear tiers. The orientation of the microvilli is parallel to
0° (=dorso-ventral axis) in R4, 7 and 8; 90° in R1, 5 and 6;
and 45° in R2 and R3.
Figure 5c is a transverse paraffin section labeled with
probes specific to NcUV mRNA (black) and NcB mRNA
(red). Clearly each ommatidium contains one photoreceptor
labeled with either of these probes. The section in Fig. 5d
is labeled with probes specific to NcB mRNA (black) and
NcL mRNA (red). The less-stained area in the center of
each ommatidium corresponds to the rhabdom, while those
in the periphery correspond to photoreceptor nuclei (see
inset). The ommatidium shown in the inset has its rhabdom
completely surrounded by several NcL-containing photo-
receptors and one NcB-containing photoreceptor. Those
without the NcB labeling have a gap (Fig. 5d), which must
represent an NcUV-containing cell. By comparing the elec-
tron micrograph and the in situ hybridization results, the
NcUV- or NcB-containing photoreceptor appears to be
R1, which bears horizontally arranged microvilli, but this
remains to be confirmed by intracellular recording followed
by dye injection. Taken together, these observations indi-
cate that the compound eye of N. cincticeps consists of two
types of ommatidia: UV-L and B-L types. We counted the
number of each type of ommatidium in in situ hybridiza-
tion samples of four individuals. Of 752 ommatidia in total,
339 of them were the UV-L type (0.45 ± 0.02), and the
remainder was the B-L type (0.55 ± 0.02); the frequencies
appear to be approximately equal.
Discussion
Physiological origin of near IR sensitivity
Stimulated by a preliminary observation that N. cincticeps
leafhoppers are attracted to 735 nm light—a wavelength
supposedly too long for any insect to perceive—we initi-
ated a study of the visual system of this species. We first
measured the leafhopper’s action spectrum in the wave-
length range from 480 to 740 nm, using seven carefully cal-
ibrated monochromatic lights with various intensities. The
leafhoppers exhibit positive phototaxis at all wavelengths,
including 740 nm, with peak sensitivity at 520 nm. Individ-
uals whose compound eyes were occluded with black paint
exhibited no phototaxis, indicating that the compound eyes
are crucial for the behavior.
The phototaxis to long-wavelength light implied the
existence of red receptors in the retina of N. cincticeps.
We tested three hypotheses for how the near IR sensitiv-
ity could come about. The first possibility is the existence
of multiple L opsins as in, for example, Papilio butterflies
(Kitamoto et al. 1998; Arikawa et al. 1999). The second
possibility is a filtering effect of red pigment surrounding
Fig. 4 Phylogeny of insect opsins using the neighbor joining method.
The opsin of an octopus was used as the out-group. Nephotettix cinc-
ticeps express three opsins, NcUV (AB761158), NcB (AB761157),
NcL (AB761156), clustering with the short wavelength (S or UV),
middle wavelength (M or B) and long-wavelength (L) opsins, respec-
tively. Numbers indicate reliabilities (%) based on 1,000 bootstrap
replicates
533J Comp Physiol A (2014) 200:527–536
1 3
the rhabdom as in Pieris rapae (Wakakuwa et al. 2004) or
Colias erate (Ogawa et al. 2013). The third possibility is
neither of the above cases: the near IR sensitivity should be
attributed to the tail sensitivity of “green” receptors.
To test the first hypothesis, we carried out RT-PCR,
and thus identified three opsins, NcL, NcB and NcUV, in
the eye of N. cincticeps (Fig. 4). N. cincticeps is a species
belonging to the family Cicadellidae (superfamily Mem-
bracoidea). We also investigated three closely related plan-
thopper species, Nilaparvata lugens, Sogatella furcifera,
Laodelphax striatellus, which belong to the family Delpha-
cidae (superfamily Fulgoroidea). Unlike Nephotettix, the
Delphacidae species have one L opsin and two UV opsins,
but no B opsin (Matsumoto et al. 2014). At any rate, we
have not detected any sign of duplication of L opsins in
these planthopper species. Because the ERG-determined
spectral sensitivity is well reproduced by a combination of
R354 (visual pigment with the peak absorbance at 354 nm),
R449 and R527 (Fig. 3), the NcL visual pigment is most
likely green—rather than red—absorbing.
To test the second hypothesis, we investigated eye anat-
omy. Even with a green-absorbing visual pigment, com-
pound eye systems can produce red receptors via the use
of spectral screening pigments located in the photorecep-
tors near the rhabdom. Clear examples are found in pierid
butterflies. For example, the eyes of the small white, Pieris
rapae, contain 620 nm-peaking red receptors and 640 nm-
peaking deep red receptors, both expressing PrL, a 563 nm-
absorbing visual pigment (Wakakuwa et al. 2004). The red
and deep red receptors are located in the proximal tier of
ommatidia whose rhabdoms are surrounded by pigment
that is red and deep red in color, respectively. The PrL
opsin is also expressed in some photoreceptors in the distal
tier, which are all green sensitive. What makes the “green
receptors” in the proximal tier into red receptors is the fil-
tering effect of the reddish pigment granules surrounding
the rhabdom. The granules act as a spectral filter because
they are positioned close to the rhabdom perimeter, where
they absorb the boundary wave propagating outside the
light-guiding rhabdom (Nilsson et al. 1988; Stavenga and
Arikawa 2011). However, if the pigment granules are
located at a distance from the rhabdom perimeter, their fil-
tering effect becomes negligible.
In the N. cincticeps ommatidia, the photoreceptor cell
body is filled with many black pigment granules. The pig-
ment functions to optically isolate the ommatidia from its
neighbors by absorbing stray light. However, they do not
absorb the boundary wave, because they are pushed away
from the rhabdom perimeter by an array of smooth-sur-
faced endoplasmic reticulum. The structure, often called
Fig. 5 Structure of the omma-
tidium of N. cincticeps. Upper
and lower edges of pictures
correspond to the dorsal and
the ventral sides of the eye,
respectively. a Azur II stained
transverse section. Each omma-
tidium is surrounded by pig-
ment granules (scale 10 μm). b
Electron micrograph of a rhab-
dom (Rh). Each ommatidium
contains eight photoreceptor
cells (R1-R8). Belt desmosomes
(arrows) indicate borders
between photoreceptors. P,
pigment granule (scale 1 μm). c
Transverse section labeled with
the NcUV probe (black) and the
NcB probe (red) (scale 10 μm;
same in d. d Transverse section
labeled with the NcB probe
(black) and the NcL probe
(red). Inset an ommatidium
showing the rhabdom (Rh) and
a photoreceptor nucleus (n). e
Ommatidial heterogeneity in
bees and butterflies (3 types)
and in leafhoppers (2 types)
534 J Comp Physiol A (2014) 200:527–536
1 3
the palisade, may even function as a spectrally flat mirror
(Horridge and Barnard 1965; Arikawa et al. 1987b). Fig-
ure 5a and b were taken from an individual that was moder-
ately light adapted with indoor light. With increasing light
adaption, the palisade may become reduced, allowing the
pigment granules to come close to the rhabdom perimeter,
as observed in a locust (Williams 1982). The pigment gran-
ules then may start to absorb the boundary wave and thus
reduce the light flux. However, the black color of the pig-
ment granules makes it unlikely that they act as a spectral
filter like the red pigment in the eyes of Pieris and Colias
(Arikawa et al. 2009). The photoreceptors of N. cincticeps
that express the NcL visual pigment therefore must be
green receptors.
Because neither the first nor the second possibility is
supported, it is not likely that the eyes of N. cincticeps are
furnished with red receptors as demonstrated in other insect
species. We therefore conclude that the third hypothesis
holds: the near IR sensitivity must be attributed to expo-
nentially attenuating residual spectral sensitivity of green
receptors in the long-wavelength region (Griffin et al.
1947; Goldsmith 1965; Lamb 1995; Govardovskii et al.
2000). The correspondence of the action spectrum with the
eye’s spectral sensitivity is consistent with this conclusion
(Fig. 3b).
Ommatidial heterogeneity and possible color vision
In situ hybridization has revealed that all ommatidia of N.
cincticeps contain a photoreceptor, designated R1, which is
labeled with either a probe specific to the mRNA of NcUV,
or NcB. The other seven photoreceptors, R2-R8, are all
labeled with the NcL probe (Fig. 5c–e). This means that the
eye of N. cincticeps is composed of two types of omma-
tidia, which are found in approximately equal proportion.
Ommatidial heterogeneity was first identified by optical
methods in butterflies (Bernard and Miller 1970; Stavenga
2002), flies (Franceschini et al. 1981) and the common
backswimmer, Notonecta glauca (Schwind et al. 1984),
and by anatomy in a wasp (Ribi 1978). Recent investiga-
tions combining electrophysiological, anatomical and
molecular biological methods, mainly in butterflies and
bees, have revealed that their compound eyes are comprised
of three types of spectrally distinct ommatidia. The omma-
tidia of these flower-visiting species have nine photorecep-
tor cells. In the simplest case, two of nine photoreceptors
express either S or M opsin, while the other seven cells all
express an L opsin (Fig. 5e). The ommatidial heterogeneity
is thus determined by the combination of the S and M cells;
S + M, S + S, or M + M (Briscoe et al. 2003; Wakakuwa
et al. 2005). While the scheme is not identical across spe-
cies, all cases investigated to date appear to be variations
on this basic pattern (Matsushita et al. 2012).
It is of particular interest to note that three out of nine
photoreceptor cells—the S, M and one L cell—project their
axons directly to the second optic neuropil, the medulla,
while the axons of the other six L cells terminate in the
first optic neuropil, the lamina (Takemura et al. 2005). An
ommatidium of N. cincticeps contains eight photoreceptor
cells, as in locusts, dragonflies and in flies (Hardie 1985),
where two photoreceptors have long axons projecting to
the medulla (Armett-Kibel et al. 1977; Nowel and Shelton
1981; Hardie 1985). The axonal projection appears to be
correlated with the opsin they express: in general, the axons
of photoreceptors expressing S and M opsins are long (Frie-
drich et al. 2011). In flies, six of the eight photoreceptors,
the peripheral R1–6 photoreceptors, have the same broad-
band spectral sensitivity and short axons terminating in the
lamina. The other two photoreceptors, R7 and R8, have vari-
able spectral sensitivities and long axons. The fly ommatidia
are divided into two types according to the spectral sensi-
tivities of R7 and R8: they are UV and B sensitive in one
type, while they are UV and G sensitive in the other type.
Because of this spectral heterogeneity, the photoreceptors
with long axons are thought to provide crucial input to the
color vision system (Strausfeld and Lee 1991; Kelber and
Henze 2013; Schnaitmann et al. 2013). Although the anat-
omy of photoreceptor axons is not known in N. cincticeps,
it is most likely that the photoreceptors with long axons are
the S or M opsin-expressing R1 and one of the L opsin-
expressing cells (R2–8). Presumably, these two photorecep-
tors form the basis of a trichromatic color vision system.
Perspectives
Our search for red receptors in a hemipteran species, whose
eye structure and function have not been explored in detail,
somewhat unexpectedly demonstrated that extreme red
sensitivity can be mediated by green receptors. This opens
up a new horizon in the study of insect vision where the
sensitivity to long-wavelength light has been largely under-
estimated. The action spectra of phototaxis have been
measured not only to answer purely biological questions
(Bertholf 1931b, 1932; Schümperli 1973; Hu and Stark
1977), but also to obtain insights into how to control pests
(Hollingsworth et al. 1964; Green 1985; Reisenman and
Lazzari 2006). In none of these previous studies was light
of wavelengths >700 nm used, probably because of the pre-
conception that insects are blind in this wavelength region.
However, as we have shown here, leafhoppers can indeed
detect near IR light (~740 nm) and are clearly attracted to
the light source. Consequently, near IR sensitivity may be
found to be widespread among insects. This could have
important applications in the control of certain pests,
because near IR light is neither harmful nor (clearly) vis-
ible to humans.
535J Comp Physiol A (2014) 200:527–536
1 3
Acknowledgments We thank Dr. Doekele Stavenga for criti-
cal reading the manuscript. This study was supported by the MAFF
(Ministry of Agriculture, Forestry and Fisheries of Japan) grant no.
INSECT-1101 to K.A. All experiments were conducted according to
the MEXT (Ministry of Education, Culture, Sports, Science and Tech-
nology of Japan) guidelines for proper conduct of animal experiment
and related activities in academic research institutions.
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